U.S. patent application number 15/112886 was filed with the patent office on 2016-12-01 for inhibitors of sterol metabolism for their use to accumulate triglycerides in microalgae, and methods thereof.
The applicant listed for this patent is CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE, COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES. Invention is credited to Caroline Barette, Jean-Christophe Cintrat, Melissa Conte, Lina-Juana Dolch, Denis Falconet, Juliette Jouhet, Eric Marechal, Coline Mei, Dimitris Petroutsos, Fabrice Rebeille.
Application Number | 20160348138 15/112886 |
Document ID | / |
Family ID | 50189641 |
Filed Date | 2016-12-01 |
United States Patent
Application |
20160348138 |
Kind Code |
A1 |
Conte; Melissa ; et
al. |
December 1, 2016 |
INHIBITORS OF STEROL METABOLISM FOR THEIR USE TO ACCUMULATE
TRIGLYCERIDES IN MICROALGAE, AND METHODS THEREOF
Abstract
The invention relates to a method for accumulating
triacylglycerols in microalgae by inhibiting the sterol metabolism,
N by incubating the microalgae with an inhibitor of sterol
metabolism. The invention also relates to a method for producing
fatty acids, biofuels, pharmaceutical or cosmetic compositions, and
also food supplements, comprising a triacylglycerols accumulation
step in microalgae according to the invention. Finally, the
invention concerns the use of an inhibitor of sterol metabolism to
accumulate triglycerides in microorganisms, and preferably
microalgae.
Inventors: |
Conte; Melissa; (Grenoble,
FR) ; Dolch; Lina-Juana; (Grenoble, FR) ; Mei;
Coline; (Grenoble, FR) ; Barette; Caroline;
(Sassenage, FR) ; Petroutsos; Dimitris; (Grenoble,
FR) ; Falconet; Denis; (Eybens, FR) ; Jouhet;
Juliette; (Seyssinet, FR) ; Rebeille; Fabrice;
(Voreppe, FR) ; Cintrat; Jean-Christophe; (Igny,
FR) ; Marechal; Eric; (Grenoble, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE |
Paris
Paris |
|
FR
FR |
|
|
Family ID: |
50189641 |
Appl. No.: |
15/112886 |
Filed: |
January 27, 2015 |
PCT Filed: |
January 27, 2015 |
PCT NO: |
PCT/IB2015/050614 |
371 Date: |
July 20, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C10L 1/02 20130101; Y02E
50/10 20130101; C12P 7/6463 20130101; C10L 2290/544 20130101; C10L
2200/0469 20130101; A23L 33/12 20160801; C10L 2290/26 20130101;
C12P 7/649 20130101; A23V 2002/00 20130101; Y02E 50/13
20130101 |
International
Class: |
C12P 7/64 20060101
C12P007/64; A23L 33/12 20060101 A23L033/12; C10L 1/02 20060101
C10L001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 27, 2014 |
EP |
14305111.8 |
Claims
1. A method for triggering triacylglycerols accumulation in
microalgae by inhibiting the sterol metabolism, by incubating the
microalgae with an inhibitor of sterol metabolism.
2. A method according to claim 1, wherein the incubation step is
implemented in a nitrogen medium.
3. A method according to claim 1, wherein the microalgae is
selected from the group consisting of microalgae of the diatom
phylum, the Chromalveolata phylum, and the Archaeplastidae
phylum.
4. A method according to claim 3, wherein the microalgae is
selected from the diatom micro-algae species Phaeodactylum
tricomutum and Thalassiosira pseudonana, the Chromalveolata
micro-algae species Nannochloropsis, and the Archaeplastidae
micro-algae species Chlamydomonas, Ostreococcus, and Chlorella.
5. A method according to claim 1, wherein the inhibitor of sterol
metabolism is a compound of formula (I) or a salt thereof:
##STR00012## wherein: R.sub.41 and R.sub.42, identical or
different, represent a hydrogen atom, alkyl, alkenyl, alkynyl, or
hydroxyl, --COR.sub.4a or --COOR.sub.4a group, in which R.sub.4a
represents a hydrogen atom, a linear or branched alkyl, aryl,
heteroaryl group, optionally substituted with one or more groups
independently selected from alkyl or cycloalkyl groups, or R.sub.41
and R.sub.42 form together an oxygen atom attached by a double
bond; R.sub.43 represents a hydrogen atom, or an alkyl group; and
R.sub.44, R.sub.45 and R.sub.46, identical or different, represent
a hydrogen atom, an alkyl, alkoxy, hydroxyl group, or an oxygen
atom attached by a double bond, said alkyl group being optionally
substituted with one or more halogen atoms, and including
optionally in its chain one or more sulfoxide functions.
6. A method according to claim 1, wherein the inhibitor of sterol
metabolism is a compound of formula (II): ##STR00013## wherein;
W.sub.3, X.sub.3, Y.sub.3 and Z.sub.3 represent carbon, sulphur,
nitrogen or oxygen atom; n.sub.3 and n.sub.3', independently, are
integer equal to 0 or 1 R.sub.31 and R.sub.32, identical or
different, represent a hydrogen atom, linear or branched alkyl,
alkenyl, alkynyl, alkylalkenyl, alkynylalkenyl, cycloalkyl,
alkylaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroalkyl,
heteroaryl groups, or form together a cycloalkyl group comprising 5
to 6 carbon atoms, one or two carbon atoms of said cycloalkyl group
being optionally replaced by one or two heteroatoms, or one of
R.sub.31 or R.sub.32 form together with R.sub.39 a cycloalkyl group
comprising 5 to 6 carbon atoms, one or two carbon atoms of said
cycloalkyl group being possibly replaced by one or two heteroatoms,
said R.sub.31 or R.sub.32 being optionally substituted with one or
more groups independently selected from linear or branched alkyl,
cycloalkyl), ##STR00014## alkynyl, arylalkyl, aryl, heteroaryl,
hydroxyl, halogen, nitro, --COR.sub.3a or --NR.sub.3aR.sub.3b
group, in which R.sub.3a and R.sub.3b, identical or different,
represent a hydrogen atom, or a linear or branched alkyl chain;
R.sub.33 represents a hydrogen atom, a linear or branched alkyl
chain, or a nitrile group; R.sub.34, R.sub.35, R.sub.36, R.sub.37,
R.sub.38 and R.sub.39, identical or different, represent hydrogen
or halogen atoms, or hydroxyl groups.
7. A method according to claim 1, wherein the inhibitor of sterol
metabolism is a compound of formula (III): ##STR00015## wherein:
n.sub.1 ranges from 1 to 12, R.sub.11 represents a hydrogen atom; a
--COR.sub.1a group, in which R.sub.1a is a group selected from a
linear or branched alkyl chain, optionally substituted with one or
more groups independently selected from hydroxyl, halogen, nitro,
optionally substituted benzyl groups, or optionally substituted
aryl groups such as phenyl groups, said aryl groups being
eventually substituted with one or more groups independently
selected from halogen atoms and methyl groups; R.sub.12, R.sub.13
and R.sub.14, identical or different, represent a hydrogen atom; a
linear or branched alkyl chain; a hydroxyl group; a --CH.sub.2OH
group; a --COOR.sub.1b group, in which R.sub.1b represents a
hydrogen atom, or a linear or branched alkyl chain; a
--OsiR.sub.1cR.sub.1dR.sub.1e group, in which R.sub.1c, R.sub.1d
and R.sub.1e, identical or different, represent a hydrogen atom, or
a linear or branched alkyl chain; or R.sub.12 and R.sub.13 form
together an exo methylene group; R.sub.15 represents a hydroxyl; a
--OSiR.sub.1cR.sub.1dR.sub.1e group; a --COOR.sub.1f group, in
which R.sub.1f represents a hydrogen atom, or a linear or branched
alkyl chain; a --OCOR.sub.1g group, in which R.sub.1g represents a
linear or branched alkyl chain; or R.sub.15 is a carbon forming an
ethylenic unsaturation with the tetrahydropyranone ring,
8. A method according to claim 1, wherein the inhibitor of sterol
metabolism is selected from the group consisting of
Ethinylestradiol, Estrone, Butenafine, Mevastatin, Simvastatin, and
Terbinafine.
9. A method for producing fatty acids comprising a triggering of
triacylglycerols accumulation step in microalgae as defined
according to claim 1.
10. A method for producing biofuels comprising the following steps;
(i) a triggering of triacylglycerols accumulation step in
microalgae as defined according to claim 1, followed by (ii) an
extraction step of the triacylglycerols accumulated in microalgae
during step (i), and (iii) a trans-esterification step of the
triacylglycerols recovered during step (ii).
11. A method for producing pharmaceutical or cosmetic compositions
comprising the following steps: (i') a triggering of
triacylglycerols accumulation step in microalgae as defined
according to claim 1, followed by (ii') an extraction step of the
triacylglycerols accumulated in microalgae during step (i'), and
(iii') a step of adding at least one pharmaceutically or
cosmetically acceptable excipient to the triacylglycerols recovered
during step (ii').
12. A method for producing food supplements comprising the
following steps: (I'') a triggering of triacylglycerols
accumulation step in microalgae as defined according to claim 1,
followed by (ii'') an extraction step of the triacylglycerols
accumulated in microalgae during step (i''), and (iii'') a step of
adding at least one food additive to the triacylglycerols recovered
during step (ii'').
13. The use of an inhibitor of sterol metabolism to accumulate
triglycerides in microorganism.
14. The use according to claim 13, wherein the inhibitor of sterol
metabolism is chosen from the compounds of formula (I), (II) and
(III), as defined in claim 6.
15. The use according to claim 14, wherein the inhibitor of sterol
metabolism is selected from the group consisting of
Ethinylestradiol, Estrone, Butenafine, Mevastatin, Simvastatin,
Terbinafine, and combinations thereof.
16. A method according to claim 3, wherein the microalgae is
selected from the group consisting of Nannochloropsis gaditana,
Nannochloropsis oceanica, and Nannochloropsis salina.
17. A method according to claim 6, wherein in the compound of
formula (II): ##STR00016## n.sub.3 and n.sub.3' are equal to 1.
Description
[0001] The invention relates to a method for accumulating
triacylglycerols in microalgae by inhibiting the sterol metabolism.
The invention also relates to a method for producing fatty acids,
biofuels, pharmaceutical or cosmetic compositions, and also food
supplements, comprising a triacylglycerols accumulation step in
microalgae according to the invention. Finally, the invention
concerns the use of an inhibitor of sterol metabolism to accumulate
triglycerides in microorganisms, and preferably microalgae.
[0002] It is acknowledged that oilseed production from crops cannot
be diverted from nutritional purpose (Durrett et al., The Plant
Journal (2008) 54, 593-607). Therefore, efforts are directed
towards the oil production in other organisms like algae (Chisti,
Biotechnology Advances 25 (2007) 294-306; Chisti, Trends in
Biotechnology, 2008, Vol 26, No. 3; Dismukes et al., Current
Opinion in Biotechnology 2008, 19:235-240; Scott et al., Current
Opinion in Biotechnology 2010, 21:277-286; Singh et al.,
Bioresource Technology 102 (2011) 26-34). Studies undergone on
triacylglycerol (TAG, also called oil) production in algae (Table 1
below) have focused on the increase of TAG in cytosolic
droplets.
TABLE-US-00001 TABLE 1 Oil content of some algae (from Chisti,
Biotechnology Advances 25 (2007) 294-306) Microalga Oil content (%
dry wt) Botryococcus braunii 25-75 Chlorella sp. 28-32
Crypthecodinium cohnii 20 Cylindrotheca sp. 16-37 Dunaliella
primolecta 23 Isochrysis sp. 25-33 Monallanthus salina >20
Nannochloris sp. 20-35 Nannochloropsis sp. 31-68 Neochloris
oleoabundans 35-54 Nitzschia sp. 45-47 Phaeodactylum tricornutum
20-30 Schizochytrium sp. 50-77 Tetraselmis sueica 15-23
[0003] The advantages of microalgae over land plants have been
summarized in the EPOBIO report (Micro- and macro-algae: utility
for industrial application, September 2007, Editor: Dianna Bowles).
Both plants (crops) cultivable on arable lands and microalgae grown
in open ponds or in confined reactors are potential sources of TAG
and fatty acids for industrial purposes and biofuels (Dismukes et
al., Current Opinion in Biotechnology 2008, 19:235-240).
[0004] However, serious concerns have been raised by the intensive
agricultural practices the use of crops implies and the diversion
of crops from food to non-food chains. Efforts are thus needed to
develop novel generation biofuels based on photosynthetic
microorganisms.
[0005] The main advantages of microalgae in relation to plants for
the production of TAG are the following: [0006] This bioresource
does not compete with the agro-resources used for animal or human
nutrition. [0007] Algal growth can be monitored in controlled and
confined conditions in an environmental friendly process using
recycled inorganic and organic wastes generated by other human
activities and the use of microalgae allows trapping and converting
industrial byproduct gases (e.g. CO.sub.2) into valuable organic
molecules (Chisti, Biotechnology Advances 25 (2007) 294-306;
Chisti, Trends in Biotechnology, 2008, Vol 26, No. 3; Chisti,
Journal of Biotechnology 167 (2013) 201-214).
[0008] The algal biomass productivity is high, microalgae showing a
very high potential of productivity with cost savings when compared
to land plants (see Table 2). Their yield is variable and
determined by the culturing approach employed: it is relatively low
in open pond systems while it can be significantly increased in
closed photobioreactors where culture parameters can be controlled.
[0009] This bioresource does not depend on a geographical location,
or on a season.
TABLE-US-00002 [0009] TABLE 2 Comparison of biomass productivity of
major crops ("C3" or "C4" type photosynthesis) and microalgae
(extract from the EPOBIO project report, University of York
(September 2007), Table 4). "C4" crops (sorghum, "C3" crops Micro-
maize, (wheat, algae sugarcane . . . ) sunflower . . . ) Maximal
productivity (T ha.sup.-1 y.sup.-1) Microalgae (photobioreactors)
130 to 150 -- -- Higher plants -- 72 30 (maximum productivity)
Average productivity in production systems (T ha.sup.-1 y.sup.-1)
Microalgae (large scale) 10 to 50 -- -- Higher plants (field) -- 10
to 30 8 to 18 Biomass production costs 0.4-40 0.04 0.04 (USD
kg.sup.-1)
[0010] The economic viability of this sector of bioindustry is
challenged by the current limitation to combine the overall biomass
yield, i.e. dry weight of algal organic matter produced per liter
and the proportion of valuable molecules, i.e. sufficiently high
proportion of TAG per dry weight for industrial extraction and
processing (Chisti, Biotechnology Advances 25 (2007) 294-306;
Chisti, Trends in Biotechnology, 2008, Vol 26, No. 3; Chisti,
Journal of Biotechnology 167 (2013) 201-214).
[0011] In particular, the lipid composition of microalgae is
compatible with biodiesel production (Dismukes et al., Current
Opinion in Biotechnology 2008, 19:235-240; Scott et al., Current
Opinion in Biotechnology 2010, 21:277-286). The rationale for
producing biodiesel from microalgae is to use sunlight to convert
water and carbon dioxide into biomass. This biomass is then
specifically redirected towards the synthesis of oil for the
generation biofuels, by applying external stimuli like nutrient
stresses, and/or by genetic engineering of metabolism (Dorval
Courchesne et al., Journal of Biotechnology 141 (2009) 31-41).
[0012] The three most important classes of micro-algae in terms of
abundance are the diatoms (Bacillariophyceae), the green algae
(Chlorophyceae), and the golden algae (Chrysophyceae) (EPOBIO
definition). Diatoms are a major phylum of the phytoplankton
biodiversity in oceans, fresh water and various soil habitats. They
are responsible for up to 25% of the global primary productivity.
Study of this group of eukaryotes has benefited from recent
developments on Phaeodactylum tricornutum, a model of pennate
diatoms. Diatoms, like other microalgae, are considered a plausible
alternative source of hydrocarbons to replace fossil fuels or
chemicals from petrochemistry, with the advantage of having a
neutral CO.sub.2 balance, based on the hypotheses that CO.sub.2 and
water can be efficiently converted into biomass by photosynthesis
and that the carbon metabolism could be controlled so that they
accumulate energetically-rich TAG. Different phytoplanktonic
organisms of the Chromalevolata superphylum have focused the
attention for their ability to accumulate TAG, with promising
initial yields and appropriate robustness and physical properties
to be implemented in an industrial process, including Phaeodactylum
tricornutum. Phaeodaetylum tricornutum is currently used for the
industrial production of omega-3 polyunsaturated fatty acids but
industrial implementation for this application and for other
applications such as biofuels is still limited by the growth
retardation and low yield in biomass when TAG accumulation is
triggered using conventional nutrient starvation approaches, such
as nitrogen starvation (Chisti, Journal of Biotechnology 167 (2013)
201-214). These approaches have an important drawback which is the
limitation of growth that the nitrogen starvation induces.
Phaeodactylum tricornutum exhibits interesting properties for an
industrial implementation, like the ability to grow in the absence
of silicon or the sedimentation of cells that could be useful for
harvesting techniques. Attempts to promote TAG accumulation can
rely on various strategies that can be combined, including the
stimulation of fatty acid and TAG biosynthesis, the blocking of
pathways that diverts carbon to alternative metabolic routes and
eventually the arrest of TAG catabolism. Small molecules could act
on each of these three aspects of TAG metabolism.
[0013] It is possible to promote the accumulation of oil in
microorganisms by inhibiting or blocking metabolic pathways that
direct the carbon fluxes to alternative metabolites. For instance,
it is well known that blocking the accumulation of carbohydrate in
storage sugars such as starch, promotes the accumulation of oils
(Siaut et al., BMC Biotechnology 2011, 11:7).
[0014] However, there remains a need for alternative methods to
trigger the accumulation of oil when algae are grown in a
nitrogen-rich medium, and by trying to avoid blocking carbohydrate
storage metabolism. Indeed, the carbohydrates produced after
CO.sub.2 photosynthetic conversion serve as a source of carbon for
all other organic molecules within the cell, so blocking their
storage has a very strong negative impact on cell growth. Other
metabolic pathways using carbon might be blocked and allow a
redirection of carbon metabolism towards TAG metabolism.
[0015] In this aim, the Inventors have now identified that the
metabolism of sterols is an alternative sink of carbon, its
inhibition in microalgae triggering the accumulation of oils.
[0016] Therefore, a first subject of the invention is a method for
triggering TAG accumulation in microalgae by inhibiting the sterol
metabolism, preferably by inhibiting the synthesis of the sterols,
said method overcoming the disadvantages listed above by incubating
the microalgae with an inhibitor of sterol metabolism.
[0017] Within the framework of the invention, the term microalgae
refers to microalgae for eukaryotes.
[0018] Also in the sense of the present invention, the TAG is built
by esterification of a 3-carbon glycerol backbone at positions 1, 2
and 3 by fatty acids. Below, TAG is synthesized by esterification
of a glycerol backbone by three fatty acids (R.sub.1, R.sub.2,
R.sub.3).
##STR00001##
[0019] In the sense of the present invention: [0020] Alkyl groups
are chosen among (C.sub.1-C.sub.26)alkyl groups, preferably
(C.sub.1-C.sub.18)alkyl groups, and more preferably
(C.sub.1-C.sub.6)alkyl groups such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, tert-butyl and isobutyl radicals;
[0021] Alkenyl groups are chosen among hydrocarbon chains of 2 to
26 carbon atoms, preferably 2 to 18, and more preferably 1 to 6,
having at least one carbon-carbon double bond. Examples of alkenyl
groups include ethenyl, propenyl, isopropenyl, 2,4-pentadienyl;
[0022] Alkynyl groups are chosen among hydrocarbon chains of 2 to
26 carbon atoms, preferably 2 to 18, and more preferably 1 to 6,
having at least one carbon-carbon triple bond; [0023] Alkylalkenyl
means any group derived from an alkenyl group as defined above
wherein a hydrogen atom is replaced by an alkyl group; [0024]
Alkynylalkenyl means any group derived from an alkenyl group as
defined above wherein a hydrogen atom is replaced by an alkynyl
group; [0025] Cycloalkyl groups refer to a monovalent cyclic
hydrocarbon radical preferably of 3 to 7 ring carbons. The
cycloalkyl group can have one or more double bonds and can
optionally be substituted. The term "cycloalkyl" includes, for
examples, cyclopropyl, cyclohexyl, cyclohexenyl and the like;
[0026] Heteroalkyl groups mean alkyl groups as defined above in
which one or more hydrogen atoms to any carbon of the alkyl is
replaced by a heteroatom selected from the group consisting of N,
O, P, B, S, Si, Sb, Al, Sn, As, Se and Ge. The bond between the
carbon atom and the heteroatom may be a single or a double bond.
Suitable heteroalkyl groups include cyano, benzoyl, methoxy,
acetamide, borates, sulfones, sulfates, thianes, phosphates,
phosphonates, and the like; [0027] Alkoxy groups are chosen among
(C.sub.1-C.sub.20)alkoxy groups, and preferably
(C.sub.1-C.sub.4)alkoxy groups such as methyloxy, ethyloxy,
n-propyloxy, iso-propyloxy, n-butyloxy, sec-butyloxy, tert-butyloxy
and isobutyloxy radicals; [0028] Aryl groups means any functional
group or substituent derived from at least one simple aromatic
ring; an aromatic ring corresponding to any planar cyclic compound
having a delocalized .pi. system in which each atom of the ring
comprises a p-orbital, said p-orbitals overlapping themselves. More
specifically, the term aryl includes, but is not limited to,
phenyl, biphenyl, 1-naphthyl, 2-naphthyl, anthracyl, pyrenyl, and
the substituted forms thereof; [0029] Heteroaryl groups means any
functional group or substituent derived from at least one aromatic
ring as defined above and containing at least one hetero atom
selected from P, S, O and N. The term heteroaryl includes, but is
not limited to, furan, pyridine, pyrrole, thiophene, imidazole,
pyrazole, oxazole, isoxazole, thiazole, isothiazole, tetrazole,
pyridazole, pyridine, pyrazine, pyrimidine, pyridazine,
benzofurane, isobenzofurane, indole, isoindole, benzothiophene,
benzo[c]thiophene, benzimidazole, indazole, benzoxazole,
benzisoxazole, benzothiazole, quinoline, isoquinoline, quinoxaline,
quinazoline, cinnoline, purine and acridine. The aryl and
heteroaryl groups of the invention comprise preferably 1 to 12
carbon atoms, and more preferably 5 or 6 carbon atoms; [0030]
Arylalkyl means any group derived from an alkyl group as defined
above wherein a hydrogen atom is replaced by an aryl or a
heteroaryl group; [0031] Arylalkenyl means any group derived from
an alkenyl group as defined above wherein a hydrogen atom is
replaced by an aryl or a heteroaryl group; [0032] Arylalkynyl means
any group derived from an alkynyl group as defined above wherein a
hydrogen atom is replaced by an aryl or a heteroaryl group; [0033]
Alkylaryl means any group derived from an aryl group as defined
above wherein a hydrogen atom is replaced by an alkyl group.
[0034] According to the invention, halogen atoms are chosen among
bromine, chlorine, fluorine and iodine, and preferably bromine,
chlorine and fluorine.
[0035] The acid addition salts of the inhibitor of sterol
metabolism according to the invention may be for example chosen
among hydrochloride, hydrobromide, sulphate or bisulphate,
phosphate or hydrogenophosphate, acetate, benzoate, succinate,
fumarate, maleate, lactate, citrate, tartrate, gluconate,
methanesulphonate, benzene-sulphonate and
paratoluene-sulphonate.
[0036] According to a preferred embodiment, in the method of the
invention the inhibition of the sterol metabolism is realized by
incubating the microalgae with an inhibitor of sterol metabolism in
a nitrogen medium.
[0037] The concentration of the inhibitor of sterol metabolism may
range from 1 .mu.M to 1 M, and preferably from 5 to 20 .mu.M. The
incubation step lasts preferably from 24 to 72 hours.
[0038] The microalgae of the invention is advantageously selected
from microalgae of the diatom phylum, the Chromalveolata phylum,
and the Archaeplastidae phylum, and more advantageously from
microalgae of the diatom phylum and the Chromalveolata phylum.
Preferably, the microalgae is selected from the diatom micro-algae
species Phaeodactylum tricornutum and Thalassiosira pseudonana, the
Chromalveolata micro-algae species Nannochloropsis, and more
preferably Nannochloropsis gaditana, Nannochloropsis oceanica,
Nannochloropsis salina, and the Archaeplastidae micro-algae species
Chlamydomonas, Ostreococcus, Chlorella. More preferably, the
microalgae is selected from the diatom micro-algae species
Phaeodactylum tricornutum and Thalassiosira pseudonana, and the
Chromalveolata micro-algae species Nannochloropsis, and more
preferably Nannochloropsis gaditana, Nannochloropsis oceanica,
Nannochloropsis salina.
[0039] Sterol inhibitors consist of molecules that have an effect
on any protein activity in the biosynthesis of sterols, a pathway
that starts with the biosynthesis of HMG-CoA reductase, followed by
the biosynthesis of mevalonic acid, epoxysqualene and then all
steroid structures deriving from epoxysqualene (see FIG. 1). The
identification of compounds which inhibit sterol metabolism,
preferably with at least a decrease of 20% of total sterol content
per cell, can be achieved simply by colorimetric or fluorometric
dosage methods such as that commercialized by CellBioLabs (Total
Sterol Assay Kit, Colorimetric method, reference STA-384 or
Fluorometric method, reference STA-390), based on a treatment by
cholesterol oxidase/esterase, which has proved to be efficient on
detecting plant sterols (D. Kritchevsky and S. A. Tepper, Clinical
Chemistry, Vol. 25, No. 8, 1464-1465 (1979)), or also according to
methods such as those exemplified in the applications WO
2010/046385 and WO 97/03202.
[0040] According to an embodiment of the invention, the inhibitor
of sterol metabolism is a compound of formula (I) or a salt
thereof:
##STR00002##
wherein:
[0041] R.sub.41 and R.sub.42, identical or different, represent a
hydrogen atom, alkyl, alkenyl, alkynyl, or hydroxyl, --COR.sub.4a
or --COOR.sub.4a group, in which R.sub.4a represents a hydrogen
atom, linear or branched alkyl, aryl, heteroaryl group, optionally
substituted with one or more groups independently selected from
alkyl or cycloalkyl groups, preferably C.sub.4-C.sub.6 cycloalkyl
groups, or R.sub.41 and R.sub.42 form together an oxygen atom
attached by a double bond;
[0042] R.sub.43 represents a hydrogen atom, or an alkyl group,
preferably C.sub.1-C.sub.3 alkyl group; and
[0043] R.sub.44, R.sub.45 and R.sub.46, identical or different,
represent a hydrogen atom, alkyl, alkoxy, hydroxyl group, or an
oxygen atom attached by a double bond, said alkyl group being
optionally substituted with one or more halogen atoms, and
including optionally in its chain one or more sulfoxide
functions.
[0044] Advantageously, R.sub.41 and R.sub.42, identical or
different, represent a hydrogen atom, a nitrile, hydroxyl,
C.sub.1-C.sub.2 alkyl or --COOR.sub.4a group in which R.sub.4a is
in C.sub.1-C.sub.7 and is optionally substituted by a
C.sub.4-C.sub.6 cycloalkyl group, or R.sub.41 and R.sub.42 form
together an oxygen atom attached by a double bond.
[0045] According to another embodiment of the invention, the
inhibitor of sterol metabolism is a compound of formula (II) or a
salt thereof:
##STR00003##
wherein:
[0046] W.sub.3, X.sub.3, Y.sub.3 and Z.sub.3 represent carbon,
sulphur, nitrogen or oxygen atom, and preferably W.sub.3, X.sub.3,
Y.sub.3 and Z.sub.3 represent carbon atoms;
[0047] n.sub.3 and n.sub.3', independently, are integer equal to 0
or 1, and preferably n.sub.3 and n.sub.3' are equal to 1;
[0048] R.sub.31 and R.sub.32, identical or different, represent a
hydrogen atom, linear or branched alkyl, alkenyl, alkynyl,
alkylalkenyl, alkynylalkenyl, cycloalkyl, alkylaryl, arylalkyl, and
preferably a benzyl group, arylalkenyl, arylalkynyl, heteroalkyl,
heteroaryl groups, or form together a cycloalkyl group comprising 5
to 6 carbon atoms, one or two carbon atoms of said cycloalkyl group
being possibly replaced by one or two heteroatoms, preferably
nitrogen atoms, or one of R.sub.31 or R.sub.32 form together with
R.sub.39 a cycloalkyl group comprising 5 to 6 carbon atoms, one or
two carbon atoms of said cycloalkyl group being possibly replaced
by one or two heteroatoms, preferably oxygen atoms, said R.sub.31
or R.sub.32 being optionally substituted with one or more groups
independently selected from linear or branched alkyl, cycloalkyl
such as
##STR00004##
alkynyl, said alkynyl group being preferably a C.sub.5-C.sub.12
branched alkynyl group, arylalkyl, aryl, heteroaryl, hydroxyl,
halogen, nitro, --COR.sub.3a or --NR.sub.3aR.sub.3b group, in which
R.sub.3a and R.sub.3b, identical or different, represent a hydrogen
atom or a linear or branched alkyl chain;
[0049] R.sub.33 represents a hydrogen atom, a linear or branched
alkyl chain such as a C.sub.1-C.sub.3 alkyl group, or a nitrile
group, and preferably R.sub.33 represents a hydrogen atom;
[0050] R.sub.34, R.sub.35, R.sub.36, R.sub.37, R.sub.38 and
R.sub.39, identical or different, represent hydrogen or halogen
atoms, or hydroxyl groups, and preferably R.sub.34, R.sub.35,
R.sub.36, R.sub.37, R.sub.38 and R.sub.39 are hydrogen atoms.
[0051] The dotted lines of formula (II) represent single or double
bonds. Specifically, said W.sub.3--X.sub.3 bond or Y.sub.3--Z.sub.3
bond is single bond respectively when one of W.sub.3 or X.sub.3 of
the W.sub.3--X.sub.3 bond, or one of Y.sub.3 or Z.sub.3 of the
Y.sub.3--Z.sub.3 bond, is sulphur or oxygen. Said W.sub.3--X.sub.3
bond or Y.sub.3--Z.sub.3 bond is double bond respectively when
W.sub.3 and X.sub.3, or Y.sub.3 and Z.sub.3 are carbon or
nitrogen.
[0052] According to a preferred embodiment, R.sub.31 and R.sub.32,
identical or different, represent a hydrogen atom, linear or
branched alkyl, alkenyl, alkynyl, alkynylalkenyl, cycloalkyl,
alkylaryl, arylalkyl, and preferably a benzyl group, arylalkenyl,
arylalkynyl, heteroalkyl, heteroaryl groups, or form together a
cycloalkyl group comprising 5 to 6 carbon atoms, one or two carbon
atoms of said cycloalkyl group being possibly replaced by one or
two heteroatoms, preferably nitrogen atoms, or one of R.sub.31 or
R.sub.32 form together with R.sub.39 a cycloalkyl group comprising
5 to 6 carbon atoms, one or two carbon atoms of said cycloalkyl
group being possibly replaced by one or two heteroatoms, preferably
oxygen atoms, said R.sub.31 or R.sub.32 being optionally
substituted with one or more groups independently selected from
linear or branched alkyl, cycloalkyl such as
##STR00005##
alkynyl, said alkynyl group being preferably a C.sub.5-C.sub.12
branched alkynyl group, arylalkyl, aryl, heteroaryl, hydroxyl,
halogen, nitro, --COR.sub.3a or --NR.sub.3aR.sub.3b group, in which
R.sub.3a and R.sub.3b, identical or different, represent a hydrogen
atom or a linear or branched alkyl chain.
[0053] According to a more preferred embodiment, R.sub.31 and
R.sub.32, identical or different, represent C.sub.1-C.sub.6 linear
or branched alkyl chains, or an arylalkyl group, optionally
substituted with one or more groups independently selected from
linear or branched alkyl. Advantageously, R.sub.31 represents a
C.sub.1-C.sub.6 linear or branched alkyl chain, and R.sub.32 a
benzyl group substituted with a C.sub.1-C.sub.6 linear or branched
alkyl chain, or an alkenyl group substituted with a linear or
branched alkynyl group, such as a
--(CH.sub.2)n.sub.3''--CH.dbd.CH--C.ident.C--C(CH.sub.3).sub.3
group in which n.sub.3'' ranges from 1 to 6.
[0054] According to another embodiment of the invention, the
inhibitor of sterol metabolism is a compound of formula (III) or a
salt thereof:
##STR00006##
wherein:
[0055] n.sub.1 ranges from 1 to 12, and preferably n.sub.1=2,
[0056] R.sub.11 represents a hydrogen atom; a --COR.sub.1a group,
in which R.sub.1a is a group selected from a linear or branched
alkyl chain, optionally substituted with one or more groups
independently selected from hydroxyl, halogen, nitro, optionally
substituted benzyl groups, or optionally substituted aryl groups
such as phenyl groups, said aryl groups being eventually
substituted with one or more groups independently selected from
halogen atoms and methyl groups;
[0057] R.sub.12, R.sub.13 and R.sub.14, identical or different,
represent a hydrogen atom; a linear or branched alkyl chain; a
hydroxyl group; a --CH.sub.2OH group; a --COOR.sub.1b group, in
which R.sub.1b represents a hydrogen atom, or a linear or branched
alkyl chain; a --OSiR.sub.1cR.sub.1dR.sub.1e group, in which
R.sub.1c, R.sub.1d and R.sub.1e, identical or different, represent
a hydrogen atom, or a linear or branched alkyl chain; or R.sub.12
and R.sub.13 are fused together to form an exo methylene group;
[0058] R.sub.15 represents a hydroxyl group; a
--OSiR.sub.1cR.sub.1dR.sub.1e group as defined above; a
--COOR.sub.1f group, in which R.sub.1f represents a hydrogen atom,
or a linear or branched alkyl chain; a --OCOR.sub.1g group, in
which R.sub.1g represents a linear or branched alkyl chain,
preferably a C.sub.1-C.sub.3 alkyl chain, and more preferably a
C.sub.1 alkyl group; or R.sub.15 is a carbon forming an ethylenic
unsaturation with the tetrahydropyranone ring.
[0059] According to a preferred embodiment, R.sub.11 is a
--COR.sub.1a group, in which R.sub.1a is a linear or branched alkyl
chain, and preferably R.sub.11 is a --COCH(CH.sub.3)C.sub.2H.sub.5
or --COC(CH.sub.3).sub.2C.sub.2H.sub.5 group.
[0060] Advantageously, R.sub.14 represent a C.sub.1-C.sub.6 alkyl
chain, preferably a --CH.sub.3 group, and both R.sub.12 and
R.sub.13 represent hydrogen atoms. Alternatively, R.sub.12 and
R.sub.14 may represent a C.sub.1-C.sub.6 alkyl chain, preferably a
--CH.sub.3 group, and R.sub.13 a hydrogen atom.
[0061] According to another preferred embodiment, n.sub.1=2, and
R.sub.15 represents a hydroxyl group.
[0062] According a preferred embodiment of the invention, the
inhibitor of sterol metabolism is selected from Ethinylestradiol,
Mevastatin, Simvastatin, Butenafine, Terbinafine, Estrone. The most
preferred inhibitors of sterol metabolism of formula (I) are
Ethinylestradiol and Estrone. The most preferred inhibitor of
sterol metabolism of formula (II) is Butenafine. The most preferred
inhibitors of sterol metabolism of formula (III) are Mevastatin and
Simvastatin.
[0063] The inhibitors of sterol metabolism of the invention are all
acting on the "mevalonate" pathway, and not on the "non-mevalonate"
pathway of synthesis of sterols (see FIG. 1).
[0064] More specifically: [0065] the inhibitors of sterol
metabolism of formula (I) are acting as steroid structural analogs
interacting with protein involved in sterol metabolism including
Estrone (Merola and Arnold, Science, Vol. 144, 301-302 (1964)) and
Ethinylestradiol (Koopen et al., Journal of Lipid Research, Vol.
40, 1999), [0066] the inhibitors of sterol metabolism of formula
(II) are acting as squalene epoxidase inhibitors (Belter et al.,
Biol. Chem., Vol. 392, 1053-1075 (2011)), and [0067] the inhibitors
of sterol metabolism of formula (III) are acting as HMG-CoA
reductase inhibitors (Liu et al., Mol. Biol. Rep. (2010)
37:1391-1395).
[0068] Another subject-matter of the invention is a method for
producing fatty acids comprising a triggering of triacylglycerols
accumulation step in microalgae as defined according to the
invention, followed by an extraction step of the triacylglycerols
accumulated in the microalgae.
[0069] The invention also relates to a method for producing
biofuels comprising the following steps:
[0070] a triggering of triacylglycerols accumulation step in
microalgae as defined according to the invention, followed by
[0071] (ii) an extraction step of the triacylglycerols accumulated
in microalgae during step (i), and
[0072] (iii) a trans-esterification step of the triacylglycerols
recovered during step (ii), for example as described by Zhang et
al., Bioresource Technology 147 (2013) 59-64.
[0073] The invention also concerns a method for producing
pharmaceutical or cosmetic compositions comprising the following
steps:
[0074] (i') a triggering of triacylglycerols accumulation step in
microalgae as defined according to the invention, followed by
[0075] (ii') an extraction step of the triacylgycerols accumulated
in microalgae during step (i'), and
[0076] (iii') a step of adding at least one pharmaceutically or
cosmetically acceptable excipient to the triacylglycerols recovered
during step (ii').
[0077] The invention also relates to a method for producing human
food and animal feed supplements comprising the following
steps:
[0078] (i'') a triggering of triacylglycerols accumulation step in
microalgae as defined according to the invention, followed by
[0079] (ii'') an extraction step of the triacylglycerols
accumulated in microalgae during step (i''), and
[0080] (iii'') a step of adding at least one food additive to the
triacylglycerols recovered during step (ii'').
[0081] The methods of the invention may comprise one or more
extraction steps after the triggering of triacylglycerols
accumulation step in microalgae. The extraction step may be
implemented using solvents or another extraction method well known
form the skilled artisan.
[0082] Another subject-matter of the invention relates to the use
of an inhibitor of sterol metabolism to accumulate triglycerides in
microorganism, preferably in microalgae, more preferably in
microalgae of the diatom phylum, and still more preferably in the
diatom microalgae species Phaeodactylum tricornutum.
[0083] Advantageously, the invention concerns the use of an
inhibitor of sterol metabolism selected from the compounds of
formula (I), (II) and (III), such as Ethinylestradiol, Mevastatin,
Simvastatin, Butenafine, Terbinafine, Estrone, to accumulate
triglycerides in microorganism, preferably in microalgae, more
preferably in microalgae of the diatom phylum, and still more
preferably in the diatom microalgae species Phaeodactylum
tricornutum. The invention also concerns the use of combination of
inhibitors of sterol metabolism selected from the compounds of
formula (I), (II) and (III), such as Ethinylestradiol, Mevastatin,
Simvastatin, Butenafine, Terbinafine, Estrone, as well as their
combination with other methods known to enhance the accumulation of
TAG in microalgae, in particular a shortage of nutrient, and more
preferably a shortage of nitrogen.
[0084] In addition to the above provisions, the invention also
comprises other provisions which will emerge from the remainder of
the description which follows, and also to the appended drawings in
which:
[0085] FIG. 1 gives a schematic view of the sterol biosynthetic
pathway via mevalonic acid, and represents the action of Mevastatin
and Simvastatin (Liu et al., Mol. Biol. Rep. (2010) 37:1391-1395),
Estrone (Merola and Arnold, Science, Vol. 144, 301-302 (1964),
Butenafine and Terbinafine (Belter et al., Biol. Chem., Vol. 392,
1053-1075 (2011)),
[0086] FIG. 2 represents pictures of a Phaeodactylum cell
visualized by confocal microscopy after a 72h cultivation in
Nitrogen-rich N(+) or in Nitrogen-starved N(-) medium On the left
side (A and C), cells are shown in phase contrast ("phase"). On the
right side (B and D), cells are shown after excitation of Nile red
at 488 nm and emission at 519 nm,
[0087] FIG. 3a-h illustrates the dose response for
Ethinylestradiol, Mevastatin, Simvastatin and Estrone, and
[0088] FIG. 4 illustrates the dose response for Ethinylestradiol,
Mevastatin and Simvastatin with an evaluation of the TAG content by
staining with Nile Red and an evaluation of cells by counting in an
aliquote fraction using a Malassez cell,
[0089] FIG. 5 shows the effect of Butenafine on Nile Red
accumulation in Nannochloropsis gaditana grown respectively in ESAW
1N1P media and ESAW 10N10P media. Nile Red Fluorescence was
normalized by calculating the relative fluorescence units per
million cells, and
[0090] FIG. 6 shows the effect of Ethinylestradiol on Nile Red
accumulation in Nannochloropsis gaditana grown respectively in ESAW
1N1P media and ESAW 10N10P media. Nile Red Fluorescence was
normalized by calculating the relative fluorescence units per
million cells.
EXAMPLES
1) Materials & Methods
[0091] 1) 1-Phaeodactylum tricornutum Strain and Growth
Conditions
[0092] Phaeodactylum tricornutum (Pt1) Bohlin Strain 8.6 CCMP2561
(Culture Collection of Marine Phytoplankton, now known as NCMA:
National Center for Marine Algae and Microbiota) was used in
experiments.
[0093] Pt1 was grown at 20.degree. C. in 250 mL flask using
"enriched artificial seawater" (ESAW) medium, prepared following
the recommendations of the Canadian Center for the Culture of
Microorganisms.
[0094] To prepare the ESAW medium, four separated solutions are
prepared, two solutions of salts (solutions 1 and 2), one solution
of nutrients, and one solution of vitamins. Salts are added in
order to distilled deionized water (DDW). When the salts in
solutions 1 and 2 are completely dissolved, the solutions 1 and 2
are mixed together. The total volume is diluted with DDW.
TABLE-US-00003 TABLE 3 Compositions of the ESAW salts solutions 1
and 2 Molecular weight Amount to weight Concentration (g
mol.sup.-1) (g/L solution) (mM) Solution 1: Anhydrous salts NaCl
58.44 20.756 362.7 Na.sub.2SO.sub.4 142.04 3.477 25.0 KCl 74.56
0.587 8.03 NaHCO.sub.3 84 0.17 2.067 KBr 119.01 0.0845 0.725
H.sub.3BO.sub.3 61.83 0.022 0.372 NaF 41.99 0.0027 0.0657 Solution
2: Hydrated salts MgCl.sub.2.cndot.6H.sub.2O 203.33 9.395 47.18
CaCl.sub.2.cndot.2H.sub.2O 147.03 1.316 9.134
SrCl.sub.2.cndot.6H.sub.2O 266.64 0.0214 0.082
TABLE-US-00004 TABLE 4 Nutrient Enrichment Stocks Stock
concentration Final concentration Solutions (g L.sup.-1) (.mu.M) 1
NaNO.sub.3 46.67 549.1 2* Na.sub.2 glycerophosphate 6.67 21.8 3
Na.sub.2SiO.sub.3.cndot.9H.sub.2O 15.00 105.6 4**
Na.sub.2EDTA.cndot.2H.sub.2O 3.64 9.81
Fe(NH.sub.4).sub.2(SO.sub.4).sub.2.cndot.6H.sub.2O*** 2.34 5.97
FeCl.sub.3.cndot.6H.sub.2O 0.16 0.592 5 MnSO.sub.4.cndot.4H.sub.2O
0.54 2.42 ZnSO.sub.4.cndot.7H.sub.2O 0.073 0.254
CoSO.sub.4.cndot.7H.sub.2O 0.016 0.0569
Na.sub.2MoO.sub.4.cndot.2H.sub.2O 0.126 0.520
Na.sub.2EDTA.cndot.2H.sub.2O 1.89 5.05 6 H.sub.3BO.sub.3 3.80 61.46
7 NaSeO.sub.3 0.00173 0.001 *Na.sub.2 glycerophosphate can be
replaced with an equimolar stock of Na.sub.2HPO.sub.4.
**Na.sub.2EDTA.cndot.2H.sub.2O is added before the trace metals
Fe(NH.sub.4).sub.2(SO.sub.4).sub.2.cndot.6H.sub.2O and
FeCl.sub.3.cndot.6H.sub.2O.
***Fe(NH.sub.4).sub.2(SO.sub.4).sub.2.cndot.6H.sub.2O can be
replaced with an equimolar stock of FeCl.sub.3. Solution 5 is
adjusted to pH = 6 with 2 g of Na.sub.2CO.sub.3. Solution 4 can be
heated to dissolve the iron.
TABLE-US-00005 TABLE 5 Vitamin Stocks Stock concentration Final
concentration Vitamin Stock (g L.sup.-1) (mM) Thiamine 0.1 2.97
.times. 10.sup.-1 Vitamin B12 0.002 1.47 .times. 10.sup.-3 Biotin
0.001 4.09 .times. 10.sup.-3
[0095] To prepare the ESAW medium, the solutions are filtered
through 0.45 .mu.m membrane filter with a glass fiber prefilter. A
flask is acid-washed in 10% HCl and rinsed in distilled water
before first use. To 1 L of filtered salt solution, 1 mL of
Nutrient Enrichment Stock solutions 1, 2, 4, 5, 6 and 7, 2 mL of
Nutrient Enrichment Stock solution 3, and 2 mL of the Vitamin Stock
are added (Tables 4 and 5). To reduce precipitation during
autoclaving, 1.44 mL of 1N HCl and 0.12 g of sodium bicarbonate are
added. The obtained ESAW medium is then sterilized by
autoclaving.
[0096] Cells were grown on a 12:12 light (450 .mu.Einstein-1
sec.sup.-1)/dark cycle (an Einstein defined the energy in one mole
(6.022.times.10.sup.23) of photons). Cells were sub-cultured every
week by inoculate fresh media with 1/5 of previous culture.
Nitrogen-rich N(+) medium contained no source of nitrogen.
Nitrogen-starved N(-), medium contained 0.05 g/L NaNO.sub.3. To
monitor cell growth, a genetically modified strain containing a
Histone H4 protein fused to the yellow fluorescent protein was used
(Siaut et al., Gene 406 (2007) 23-35).
[0097] 1) 2-Principle of Nile Red Staining of Oil Droplets
[0098] Accumulation of oil droplets can be monitored by Nile Red
(Sigma Aldrich) fluorescent staining (Excitation wavelength at 485
nm; emission at 525 nm) as described by Ren et al. (Biotechnology
for Biofuels 2013, 6:143), Cells were diluted and adjusted to a
cell density that was linearly correlated with Nile Red
fluorescence. Nile Red solution (40 .mu.L of 2.5 .mu.g.mL.sup.-1
stock concentration, in 100% DMSO) was added to 160 .mu.L cell
suspension. Specific fluorescence was determined by dividing Nile
Red fluorescence intensity by the number of cells. Oil bodies
stained with Nile Red were then visualized using a Zeiss
AxioScope.A1 microscope (FITC filter; Excitation wavelength at 488
nm; emission at 519 nm).
[0099] Lipid droplets can be visualized. In FIG. 2, a Phaeodactylum
cell was visualized by confocal microscopy after a 72h cultivation
in Nitrogen-rich N(+) medium or in Nitrogen-starved N(-) medium. On
the left side, cells are shown in phase contrast ("phase"). On the
right side, cells are shown after excitation of Nile red at 488 nm
and emission at 519 nm. Lipid droplets can be clearly
visualized.
[0100] This principle was use to measure the presence of oil in
Phaeodactylum tricornutum simply by using a spectrofluorometer (in
these conditions, to lower the detection of other fluorophores
within the cell, such as chlorophylls, excitation was at 530 nm and
emission at 580 nm).
[0101] 1) 3-Alternative Method for Oil Level Detection
[0102] Alternatively, oil is extracted using solvents or another
extraction method, separated and purified by thin layer
chromatography and methanolyzed to produce fatty acid methyl esters
and quantified by gas chromatography coupled to a ionization flame
detector or a mass spectrometer.
[0103] TAG were extracted from 200 mg of freeze-dried Phaeodactylum
tricornutum cells in order to prevent lipid degradation. Briefly,
cells were frozen in liquid nitrogen immediately after harvest. The
freeze-dried cell pellet was resuspended in 4 mL of boiling ethanol
for 5 minutes followed by the addition of 2 mL of methanol and 8 mL
of chloroform at room temperature. The mixture was then saturated
with argon and stirred for 1 h at room temperature. After
filtration through glass wool, cell remains were rinsed with 3 mL
of chloroform/methanol (2:1, v/v). In order to initiate biphase
formation, 5 mL of NaCl 1% was then added to the filtrate. The
chloroform phase was dried under argon before re-solubilization of
the lipid extract in pure chloroform. To isolate TAG, lipids were
run on silica gel thin layer chromatography (TLC) plates (Merck)
with hexane/diethylether/acetic acid (70:30:1, v/v). Lipids were
then visualized under UV light after pulverization of
8-anilino-1-naphthalenesulfonic acid at 2% in methanol. They were
then scraped off from the TLC plates for further analyses. For acyl
profiling and quantification of TAG, fatty acids were methylated
using 3 mL of 2.5% H.sub.2SO.sub.4 in methanol during 1 h at
100.degree. C. (including standard amounts of 21:0). The reaction
was stopped by the addition of 3 mL of water and 3 mL of hexane.
The hexane phase was analyzed by gas liquid chromatography (Perkin
Elmer) on a BPX70 (SGE) column. Methylated fatty acids were
identified by comparison of their retention times with those of
standards and quantified by surface peak method using 21:0 for
calibration. Extraction and quantification were done at least 3
times.
[0104] 1) 4-Principle of Cell Normalization
[0105] The number of cells in a sample can be evaluated using a
fluorescent reporter, like the strain by Siaut et al. (Gene 406
(2007) 23-35) containing a genetic construction with Histone H4
protein fused to the Yellow Fluorescent Protein (YFP) (FIG. 5A of
Siaut et al., Gene 406 (2007) 23-35).
[0106] For cell counting, we can either use this strain called
"ptYFP" and measure the fluorescence emitted by the YFP at 530 nm
after excitation at 515 nm, or use any strain and estimate cell
numbers by counting with a Malassez grid (supplier:
Mareinfeld).
[0107] 1) 5-Incubation of Phaeodactylum tricornutum with Inhibitors
of the Sterol Metabolism and Detection of Oil Accumulation
Triggered by the Treatment
[0108] On the first day, we prepared 48 well plates by adding 4 mm
glass beads sterilized with Ultra-Violet exposure in each well.
[0109] We prepared fresh suspensions of microalgae in exponential
growth phase. For cell normalization based on YFP fluorescence,
cells of Phaeodactylum tricornutum containing a YFP reporter
(ptYFP) cultured in N(+) ESAW medium were centrifuged at 3,500 rpm,
5 min. For cell normalization based on counting using a Malassez
grid, cells of the Pt1 strain of Phaeodactylum tricornutum cultured
in N(+) ESAW medium were centrifuged at 3,500 rpm, 5 min. The
supernatant was discarded and the pellet suspended in N(-) ESAW.
The microalgae were then centrifuged at 3,500 rpm, 5 min. The
supernatant was discarded and the pellet suspended in N(-) ESAW.
Cells were then diluted to 1.times.10.sup.6 cells/mL in N(-) ESAW.
Samples were then separated into two batches. One was supplemented
with 1 .mu.L/mL of 46.7 g/L NaNO.sub.3 stock to obtain a suspension
of cells in N(+) ESAW medium. Another was left without NaNO.sub.3
to obtain a N(-) ESAW culture as a control for high lipid
accumulation. In a 48-well clear NUNC plate, 450 .mu.L/well of
1.times.10.sup.6 cells/mL were dispensed.
[0110] For a dose-response analysis, each well of the 48-well plate
was then subjected to an appropriate incubation with 0, 1, 10 or
100 .mu.M of inhibitor of sterol metabolism using 50 .mu.L of the
following: 50 .mu.L of a 10 time concentrated solution of inhibitor
of sterol metabolism (5% DMSO:95% N(+) ESAW) or 50 .mu.L of
Nitrogen-rich medium without any inhibitor of sterol metabolism (5%
DMSO:95% N(+) ESAW) or Nitrogen-starved medium without any
inhibitor of sterol metabolism (5% DMSO:95% N(-) ESAW) as a
positive control.
[0111] For an analysis of the effect of a single dose, an
incubation was performed with 0, and a chosen concentration of
inhibitor of sterol metabolism (50 .mu.M).
[0112] In all cases, the edge of the plate was sealed using a
parafilm. Plates were incubated for 48 h in an incubator with top
lighting, 20.degree. C., 100 rpm, 12 h/12 h light/dark.
[0113] After an incubation of 48 hours, fluorescence was measured
at the following excitation/emission wavelengths, 530/580 nm (to
evaluate a baseline fluorescence prior Nile Red addition) and
515/530 nm (to evaluate YFP fluorescence). Following this first
measure, 40 .mu.L of Nile Red (2.5 .mu.g/mL stock concentration, in
100% DMSO) are added. Plates are mixed and incubated 20 minutes at
room temperature, protected from light. Nile Red fluorescence is
then measured using a spectrofluorometer (excitation 530
nm/emission 580 nm).
[0114] 1) 6-Incubation of Nannochloropsis gaditana with Inhibitors
of the Sterol Metabolism and Detection of Oil Accumulation
Triggered by the Treatment
[0115] Experiments were performed in two different conditions of
Nannochloropsis gaditana: cells were either cultured in ESAW
containing 47 mg.L.sup.-1 NaNO.sub.3 and 3 mg.L.sup.-1
NaH.sub.2PO.sub.4 (medium 1N1P), or 470 mg/L NaNO.sub.3 and 30
mg.L.sup.-1NaH.sub.2PO.sub.4) (medium 10N10P). Cells in an
exponential growth phase were collected via centrifugation at 3500
rpm for 10 minutes. The supernatant was discarded and the cells
were resuspended in the same volume of ESAW medium (either 1N1P or
10N10P). The cultures were centrifuged again at 3500 rpm, for 10
minutes, and the supernatant was discarded. The pellet was
resuspended in ESAW (either 1N1P or 10N10P) to obtain a
concentration of 2.times.10.sup.6 cells/mL. Cell counts were
performed using a Malassez counting chamber, allowing 10 minutes
for the cells to settle before counting.
[0116] Twenty milliliters of 2.times.10.sup.6 cells/mL of
Nannochloropsis gaditana in ESAW (10N10P) and ESAW (1N1P) were
dispensed into sterile glass conical flasks. Stocks of inhibitor of
sterol metabolism were prepared in DMSO. Inhibitors of sterol
metabolism were added to the 20 mL Nannochloropsis gaditana samples
at final concentrations of 10 .mu.M, 30 .mu.M, or 100 .mu.M. The
maximum final concentration of DMSO in the samples was 1% (v/v),
All cultures were incubated for seven days at 100 rpm, 12 h/12 h
light/dark cycle, 50 .mu.E.m.sup.-2.s.sup.-1, 20.degree. C.
[0117] Each day, an aliquot was taken from each flask in order to
perform a Nile Red stain and cell counts. 160 .mu.L per sample was
added to black 96 well plates, and allowed to settle for 10
minutes. In order to detect any background noise, fluorescence was
measured at excitation and emission of 530 nm and 580 nm,
respectively. 40 .mu.L of 2.5 .mu.g.mL.sup.-1 Nile Red in DMSO was
added to each well, and mixed thoroughly. After 20 minutes of
incubation, Nile Red fluorescence was measured at excitation and
emission of 530 nm and 580 nm, respectively.
[0118] Nile Red Fluorescence was normalized by calculating the
relative fluorescence units per million cells. Results were
expressed as a percentage of Nile Red fluorescence of
Nannochloropsis gaditana cultured in complete medium (either ESAW
(10N10P) or ESAW (1N1P)).
[0119] 1) 7-Inhibitors of Sterol Metabolism
[0120] Inhibitors of sterol metabolism were obtained from the
Prestwick library for their ability to trigger the accumulation of
lipid droplets within the cells of Phaeodactylum tricornutum, and
then purchased from Sigma-Aldrich,
TABLE-US-00006 TABLE 6 Inhibitors of sterol metabolism selected
from the Prestwick library Chemical name Structure Molecular
Formula Mevastatin ##STR00007## C.sub.23H.sub.34O.sub.5 Butenafine
##STR00008## C.sub.23H.sub.27N Simvastatin ##STR00009##
C.sub.25H.sub.38O.sub.5 Estrone ##STR00010##
C.sub.18H.sub.22O.sub.2 Ethinylestradiol ##STR00011##
C.sub.20H.sub.24O.sub.2
2) Results
[0121] Phaeodactylum tricornutum was incubated for 48 h in presence
of 10 .mu.M of Mevastatin, Butenafine, Simvastatin, Estrone,
Ethinylestradiol and Terbinafine.
[0122] In all cases the presence of oil per cell increased by a
factor of at least 1.5, based on Nile Red staining.
[0123] FIG. 3a-h illustrates the dose response for
Ethinylestradiol, Mevastatin, Simvastatin and Estrone with an
evaluation of the TAG content by staining with Nile Red, and an
evaluation of cells by monitoring YFP fluorescence from an
expressed Histone H4 reporter gene. On the left, FIG. 3a-h shows
the Nile Red levels in percent of untreated cells and the number of
cells estimated by the YFP fluorescence expressed in percent of
untreated cells. The right panels show the increase of oil content
per cell, by the ratio of Nile Red fluorescence/YFP fluorescence,
expressed in percent of untreated cells. The oil content per cell
increases with drug concentration and consequently level of sterol
metabolism inhibition and ranges from 120 to 400% when compared to
untreated cells.
[0124] FIG. 4 illustrates the dose response for Ethinylestradiol
(A), Mevastatin (B) and Simvastatin (C) with an evaluation of the
TAG content by staining with Nile Red and an evaluation of cells by
counting in an aliquote fraction using a Malassez cell. In
histograms of FIG. 4, the white bars indicate the evaluation of
cell numbers in percent of untreated cells, and the black bars
indicate the Nile Red per 10.sup.6 cell, in percent of untreated
cells. The oil content per cell, and consequently level of sterol
metabolism inhibition, increases with incubation of 50 .mu.M of
inhibitor, and ranges from 120 to 350% when compared to untreated
cells.
[0125] Nannochloropsis gaditana was incubated in presence of
Butenafine and
[0126] Ethinylestradiol.
[0127] FIGS. 5 and 6 illustrate the effect of Butenafine and
Ethinylestradiol on Nile Red accumulation in Nannochloropsis
gaditana grown respectively in ESAW 1N1P media and ESAW 10N10P
media. Nile Red Fluorescence was normalized by calculating the
relative fluorescence units per million cells.
[0128] Both Butenafine and Ethinylestradiol trigger the
accumulation of oil in Nannochloropsis gaditana, in different media
and with a time course that can be observed at least for 7
days.
* * * * *